Effects of dipropylacetate on brain development.

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Effects of Dipropylacetate
on Brain Development
Jaime Diaz, PhD,' and W. Donald Shields, M D t
~
~
~~
Dipropylacetate (DPA; valproic acid) is a new anticonvulsant reported to be effective in many types of seizures,
including those that occur early in life. This study describes the effects of chronic administration of DPA upon a
developing organism. Four-day-old rat pups were injected daily with either 75 mglkg DPA, 200 mglkg DPA, or
vehicle until day 18. Administration of DPA resulted in decreased body and organ weights, with the greatest
reductions in the 200 mglkg group. To control caloric intake, an additional experiment was conducted. Animals
were injected with either vehicle or 200 mglkg DPA. Each animal was implanted with a chronic intragastric cannula and fed through the cannula. The results of this experiment indicated that body and organ weights were the
same for both vehicle and drug groups, except for brain. The animals receiving DPA had significant deficits of 12%
in total brain weight and 22% in weight of the cerebellum. The data suggest that chronic administration of DPA
early in life may have adverse consequences on brain growth.
Diaz J, Shields WD: Effects of dipropylacetate on brain development. Ann Neurol 10:465-468, 1981
Dipropylacetate (DPA; valproic acid, sodium valproate, Depakene), the most recent anticonvulsant to
be approved by the Food and Drug Administration,
has been shown to be effective in a variety of seizure
disorders [l, 17, 211. DPA was released for treatment of absence seizures, but it has also been used
extensively in infants and in young children with
other seizure types [ 15,241, including febrile convulsions [2]. Although the drug was initially considered
safe, several reports of toxicity in children attributed
to DPA treatment have described cases of acute
hepatic failure, Reye syndrome, and pancreatitis 19,
12,22,24). I n spite of its prevalent use in infants and
young children, the effects of DPA o n development
have not been examined. The present study describes
the effects of chronic administration of DPA on a developing organism, especially on development of the
brain.
Experiment 1
Materials and Methods
Four-day-old male Long-Evans hooded rats were matched
by weight and assigned to one of four groups: (1) 200 mg/
kg/day DPA (N = 8); (2) 75 mg/kg/day DPA (N = 10); (3)
a vehicle-treated control ( N = 9); and (4) a noninjected
control ( N = 9). Beginning on postnatal day 4 and continuing through day 18, the animals were injected subcutaneously each morning. Standard reflex tests (righting,
negative geotaxis, and cliff avoidance) were conducted beginning o n day 5 and on alternate days thereafter prior to
the morning injections [22]. O n day 17, all the animals
From the 'Department of Psychology (NI-25), University of
Washington, Seattle, WA 98195, and the +Department of
Neurology, UCLA School of Medicine, Los Angeles, CA 90024.
were weighed and tested in an open field [5]. The animals
were also tested in a straight-alley water maze. This test
consisted of dropping an animal at the end of a rectangular
enclosure (10 cm wide, 4 0 cm long, and 2 0 cm high) filled
with warm water. The latency for the animal to swim the
length of the maze to a ramp at the other end was recorded.
Following this test the animals were killed. The brain, liver,
spleen, and left kidney were removed and weighed.
A separate group of normally reared Long-Evans hooded
rat pups were either given daily injections of D P A (200
mg/kg) from day 4 through day 18 as just described (N =
21) o r received a single injection of DPA on day 18 (N =
21). O n day 18, pooled blood samples (3 animals per sample) were taken from both groups at 30 minutes and at 1 , 2 ,
4 , 8 , 16, and 24 hours after the D P A injection; DPA serum
levels were later determined.
Results
Neither dose of DPA produced any overt changes in
behavior. The ontogeny of reflexive behavior was not
altered in the DPA-treated groups. However, the
animals that received 200 mg/kg DPA did exhibit
motor problems, evidenced by a peculiar uncoordinated gait in the open field task. Furthermore, the
animals receiving the higher dose of DPA took
longer to swim the water maze test than the untreated
and vehicle-treated animals (mean, 18.3 seconds for
the DPA-treated animals, 7.2 seconds for the control animals; t = 3.47, df = 2 3 , p < 0.01).
Both groups receiving DPA demonstrated reductions in body and organ weights, including that of the
Received Sept 15, 1980, and in revised form Feb 18, 1981. Accepted for publication Mar 7, 1981.
Address reprint requests
fO
Dr Diaz,
0364-5134/81/110465-04$01.25 @ 1981 by the American Neurological Association
465
BODY WEIGHT ( g r a m s )
-rn
2>g
i-
140
30
Z
130
20
g 120
10
2
3
I
a
0
TOTAL B R A I N WEIGHT
(grams)
301
20
J
110
0
+
--
1000
+
960
Z
920
rn
880
m
3
3
LL
w
0
10
+
3 150
I
2
AR
CONTROL
140
AR
DPA
1
W
L
- ,160
L
,130
CEREBRUM WEIGHT ( g r a m s )
W
I
120
840
N
S
Control
7 5 200
DPA
V
13-
N
S
Control
(rng/kgl
75 200
DPA
(mglkg)
F i g 1 . Mean body and brain weights for the two control groups
(N = noninjected controls; S = vehicle-treated controls) and
the two experimentalgroups (75 = 75 mglkglday DPA; 200
= 200 mglkgiday DPA). Bars represent .ctandard error of the
mean for the individual groups.
brain (Fig l ) , when compared to the control groups.
The group given 7 5 mg/kg showed slight but
significant reductions in body weight (11%; t =
2.13, df = 26,p < 0.05) and total brain weight ( 5 % ; t
= 4.15, df = 26,p < 0.001), with a cerebral weight
decrease of 5% (t = 4.09, df = 2 6 , p < 0.001) but no
significant reductions in the weight of cerebellum,
liver, spleen, or kidney. O n the other hand, the body
and organ weights of the group receiving 200 mg/kg
of DPA were severely reduced. Compared to the
control groups, the higher-dose DPA group demonstrated a 23% diminution in body weight ( t = 4.57,
df = 24,p < 0.01) and 17% reduction in total brain
weight ( t = 14.19, df = 24,p < 0.01), with a 25%
reduction in weight of the cerebellum ( t = 9.10, df =
24, p < 0.01, Fig 1 ) and 16% reduction in cerebral
weight ( t = 14.29, df = 24,p < 0.01). T h e animals
receiving 200 mg/kg DPA also showed reductions in
liver ( 1 5 % ; t = 2.24, df = 24, p < 0.05), kidney
(16%; t = 2.72, df = 24,p < 0.02),and spleen (43%;
t = 4.93, df = 24,p < 0.01) compared to controls.
DPA serum levels peaked 30 minutes after the last
injection at 317 p,g/ml and then dropped to 18
pg/ml by 8 hours. There were no differences in
serum levels between the chronic and acute DPA
groups.
To determine if the effects seen in the DPAtreated animals may have been a result of druginduced undernutrition due to the interference of
nursing, Experiment 2 was conducted.
Experiment 2
Materials and Methods
Four-day-old Long-Evans hooded rats were matched by
weight and assigned to one of two groups: 200 mg/kg/day
466 Annals of Neurology
CEREBELLUM WEIGHT (g rams)
Vol 10 No 5
12.? 5
11-
65
10AR
AR
CONTROL D PA
AR
CONTROL
AR
DPA
F i g 2. Mean body and brain weights for the two artificially
reared (AR) groups. Bars represent standard error of the mean
for each group.
DPA (N = 5 ) , or a vehicle-treated group (N = 4). In this
experiment t h e rat pups were not returned to their mothers
but were reared using a procedure that does not require the
pups to nurse. This procedure has been previously described in detail [4, 5 , 141. Briefly, the pups were lightly
anesthetized and intragastric cannulas were permanently
implanted. The pups were housed in cups (12 cm in
diameter and 8 cm deep) that floated in a warm-water bath,
and the cannulas were connected to syringes filled with a
milk formula [18] and mounted on infusion pumps. All the
animals were infused intragastrically with enough milk
formula to maintain normal growth during the entire period of the experiment. As in Experiment l , all the animals
were weighed and injected subcutaneously each morning
from day 4 through day 18. O n day 19, all the animals were
killed and their brains, livers, kidneys, and spleens were
removed and weighed.
Results
There were no differences in body, liver, and kidney
weights between the vehicle-treated rats and those
given 200 mg/kg DPA. However, the group receiving DPA showed a significant 12% reduction in total
brain weight ( t = 3.63, df = 7 , p < 0.01) and a 21%
decrease in weight of the cerebellum ( t = 4.30, df =
7 , p < 0.01) compared to the vehicle-treated animals
(Fig 2).
Discussion
The data from the present study indicate that chronic
administration of DPA early in life may compromise
November 1981
normal growth and development, particularly that of
the brain.
A principle of pharmacological research is that
doses of a drug cannot be literally translated among
different species: some species can tolerate more or
less of a drug than others. A 2 0 0 mg/kg dose of DPA
has been shown to be effective 50% of the time in
protecting mice from picrotoxin-induced seizures
[ l l ] . Thus, the two DPA doses chosen for Experiment 1 represented one that was anticonvulsant in
rodents and one that was not, although the higher
dose did not produce accumulation of DPA in the
rats.
In Experiment 1, neither dose of DPA caused any
frank side effects. Although reflexive behavior developed normally in both drug groups, some animals
in the higher-dose DPA group displayed subtle
ataxia. These motor problems became apparent when
the animals’ coordination was challenged in the
swimming task. Compared to the lower-dose DPA
group and the control groups, the animals treated
with the higher dose of DPA exhibited severe motor
problems, reflected in the significantly longer latencies of this group in swimming to a ramp. However,
motor difficulties of any kind are not surprising,
given the 25% reduction in weight of the cerebellum
seen in this higher-dose DPA group.
The role of undernutrition in Experiment 1 is not
clear. The groups receiving DPA did have significantly lower body weights than control animals,
but their behavioral profile was not one of undernourished animals. Undernutrition is known to disrupt the ontogeny of reflexive behavior [22], yet in
this study reflexes developed normally for both drug
groups.
The question of DPA-induced undernutrition was
directly addressed in Experiment 2. The method of
intragastric feedings used in this experiment is a
powerful technique for studying early drug administration [4]. In Experiment 2, the DPA-treated animals were infused with the same amount of milkformula as the vehicle-treated animals. With caloric
intake controlled, the animals receiving DPA still had
severe brain weight deficits. In fact, the pattern of
these deficits in Experiment 2 was essentially the
same as that in Experiment 1.
The disruption of brain development by DPA
treatment is not unexpected given the rapid brain
growth that occurs during this period [ 7 ] ,especially
in the cerebellum [81. The vulnerability of the central
nervous system to a variety of exogenous insults
during this period of growth has been amply described [6, 12, 191. However, the severe reduction in
cerebellum weight seen in this study is not an invariable consequence of anticonvulsant administration
during the brain growth spurt. Previous studies [4, 51
have demonstrated that chronic administration of
phenobarbital during this same period retards brain
growth, but cerebellar growth is not retarded disproportionately to the rest of the brain. Morever, the
animals given phenobarbital did not exhibit motor
difficulties. This suggests that the unusually severe
cerebellar growth deficits and coordination problems
observed in this study may be specific to DPA and
not to anticonvulsant medication in general.
The human brain growth spurt extends well into
the fourth postnatal year [9]. During this time many
infants receive anticonvulsant medication, often for
prolonged periods. The data from the present study
demonstrate that chronic DPA treatment, at doses
which are anticonvulsant in rodents, interferes with
normal brain growth in developing rats. Even though
direct extrapolation between species can be difficult,
the data of the present study raise several critical issues which need further study.
Supported by Grant NS-07628 from the US Public Health Service, by Grants HD-05715 and HD-04612 from the National Institute of Child Health and Human Development, by Grant NS07691 from the National Institute of Neurological and Communicative Disorders and Stroke, and by the Regents of the University of California, Mental Retardation Research Center, Los
Angeles.
Presented in part at the Seventh Annual Meeting of the Child
Neurology Society, Keystone, CO, Sept 28-30, 1978.
The authors wish to thank Kathy Watanabe and Lorita Bank for
their skillful technical assistance, and the Alcoholism and Drug
Abuse Institute of the University of Washington, especially Denise Mongrain, for assistance in preparation of the manuscript.
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